Date of Award

12-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cell Biology and Biochemistry

Research Advisor

Radhakrishna Rao, Ph.D.

Committee

Polly A. Hofmann, Ph.D. Anjapravanda Naren, Ph.D. Trevor W. Sweatman, Ph.D. Christopher M. Waters, Ph.D.

Abstract

Evidence indicates that MAP kinase (ERK1/2) is involved in regulation of epithelial tight junctions. There are different opinions expressed by investigators as to whether ERK disrupts the junctions or protects them. ERK has also been demonstrated to mediate the EGF-caused protection of the intestinal epithelial tight junctions (TJ) from hydrogen peroxide. Studies using pharmacological inhibitors have shown that EGF increases Thr-phosphorylation of occludin by a MAP kinase-dependent mechanism. This study aimed at looking at the role of ERK in regulation of tight junctions using pharmacological and molecular techniques.

Hypothesis: ERK protects tight junctions in differentiated Caco-2 cells, while it is disruptive to tight junctions in under-differentiated cells, and that in differentiated cells, ERK regulates the activity of PKCζ to enhance Thr-phosphorylation of occludin.

Specific Aims: 1. To determine if MAP kinase activity induces contrasting effects in under-differentiated and differentiated intestinal epithelium and 2. To determine if ERK enhances Thr-phosphorylation of occludin by modulating the activation of PKCζ.

Technical Approach: 1. The role of ERK in EGF-mediated Thr-phosphorylation of occludin was determined by evaluating the effect of MEK inhibitor (U0126) on: a) the phosphorylation and translocation of PKCζ and PP2A, b) the association of PKCζ and PP2A in occludin immunocomplex in under-differentiated and differentiated Caco-2 cell monolayers, and c) by in vitro incubation of recombinant PKCζ and PP2A with activated ERK. Additionally, the effect of RNA interference was evaluated to determine the influence of ERK on tight junction assembly and maintenance of tight junction integrity. 2. To determine that ERK activity produces contrasting effects in under-differentiated and differentiated cells, Caco-2 cells were transfected with wild type (WT), dominant negative (DN) and constitutively active (CA) forms of MEK in an inducible vector (GFP-tagged pTRE2hyg). The expression of MEK and thus ERK was regulated in the Caco-2 cells on various stages of cellular differentiation, and its effect on tight junction integrity evaluated by measuring TER, inulin flux, immunofluorescence localization of TJ-proteins. The role of ERK in the regulation of tight junctions was also studied in mouse ileal epithelium by localization of occludin in detergent-soluble and insoluble fractions, and by immunofluorescence for tight junction proteins in the intestinal epithelial cells.

Results: Reduced expression of ERK by RNA interference enhanced the assembly and integrity of tight junctions. ERK does not directly phosphorylate occludin, but it enhances Thr-phosphorylation of PKCζ in differentiated Caco-2 cells as well as in vitroassays. PKCζ in turn, phosphorylates occludin on threonine residues. Thus ERK indirectly regulates Thr-phosphorylation of occludin, and preserves the integrity of tight junctions. ERK prevents hydrogen peroxide-induced translocation of PP2A to the TJs and thus preventing dephosphorylation of occludin by PP2A. Expression of CA-MEK in under-differentiated cells leads to disruption of tight junctions, while it enhances the tight junction integrity in differentiated cells. The opposite effects were observed with the expression of DN-MEK. In mouse ileum, ERK protected the tight junctions against oxidative stress produced by hydrogen peroxide.

Conclusions: ERK has a contrasting effect on tight junctions in differentiated and under-differentiated Caco-2 cells. This effect is produced by modulating the activation of PKCζ and translocation of both PKCζ and PP2A.

DOI

10.21007/etd.cghs.2010.0006

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